Summary: Antiretroviral Drug-Induced Changes in Synapses between Human iPSC-Derived Cortical Neurons to Assess Risk and Mechanisms of Neuropsychiatric Adverse Effects Combined antiretroviral therapy (cART) has dramatically increased the survival and improved the quality of life for HIV-1 infected patients. There is growing concern that while cART decreases HIV-1 neurotoxicity by reducing viral load, antiretroviral drugs themselves may produce neurotoxicity. In some patients, antiretroviral drugs elicit symptoms of depression and anxiety. These symptoms, like many neuropsychiatric disorders may be associated with a reduction in the number of synaptic contacts. We developed a high content imaging approach to quantify functional glutamatergic synapses between neurons in culture. This highly automated high content analysis (HCA) assay is exquisitely sensitive to agents that reduce dendritic spine density between rat neurons in primary culture. In this proposal, we describe the modification of this assay to assess synaptic contacts between human induced pluripotent stem cell (hiPSC)?derived cortical neurons. The use of human neurons is warranted because human synapses are uniquely sensitive to some neurotoxic insults. We will use this assay to evaluate the synaptic toxicity of antiretroviral drugs alone and in the presence of synaptic modifiers associated with risk of psychiatric disease. Three specific aims are proposed. In aim 1, we will optimize the HCA assay for detecting synapses between hiPSC-derived neurons. An automated assay to quantify synaptic connections between live human neurons will have broad application for studying the mechanism of synaptic changes that underlie psychiatric disorders. In aim 2, we will determine whether antiretroviral drugs are toxic to synapses between hiPSC-derived cortical neurons. These results will identify antiretroviral drugs with a particular propensity to impair synaptic function. In aim 3, we will evaluate synaptic modifiers for altered risk of antiretroviral-induced synaptic toxicity. A limited screen in the presence of increased pro-inflammatory cytokines, elevated stress hormones, or reduced levels of neurotrophic factors alone and in combination with antiretroviral drugs will be performed to determine potential synergistic effects. These studies will establish a proof of concept for using the HCA assay to identify risk factors that potentiate antiretroviral drug-induced loss of synapses between human cortical neurons. The results from this project will determine the feasibility of using an HCA assay to identify synaptic changes with the potential to produce neuropsychiatric side effects and will provide a foundation for future studies to probe the mechanism and potential reversal of synapse loss. Identification of combinations of antiretroviral drugs and risk factors that produce synapse loss could inform the clinical care of persons living with HIV. Finally, with further validation this assay may assist in developing next-generation HIV therapies by identifying potential adverse effects.